Nitrite Reductase Activity of Ferrous Nitrobindins: A Comparative Study

Nitrobindins (Nbs) are all-β-barrel heme proteins spanning from bacteria to Homo sapiens. They inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and promoting peroxynitrite isomerization to NO3−. Here, the nitrite reductase activity of Nb(II) from Mycobacterium tuberculosis (Mt-Nb(II)), Arabidopsis thaliana (At-Nb(II)), Danio rerio (Dr-Nb(II)), and Homo sapiens (Hs-Nb(II)) is reported. This activity is crucial for the in vivo production of NO, and thus for the regulation of blood pressure, being of the utmost importance for the blood supply to poorly oxygenated tissues, such as the eye retina. At pH 7.3 and 20.0 °C, the values of the second-order rate constants (i.e., kon) for the reduction of NO2− to NO and the concomitant formation of nitrosylated Mt-Nb(II), At-Nb(II), Dr-Nb(II), and Hs-Nb(II) (Nb(II)-NO) were 7.6 M−1 s−1, 9.3 M−1 s−1, 1.4 × 101 M−1 s−1, and 5.8 M−1 s−1, respectively. The values of kon increased linearly with decreasing pH, thus indicating that the NO2−-based conversion of Nb(II) to Nb(II)-NO requires the involvement of one proton. These results represent the first evidence for the NO2 reductase activity of Nbs(II), strongly supporting the view that Nbs are involved in NO metabolism. Interestingly, the nitrite reductase reactivity of all-β-barrel Nbs and of all-α-helical globins (e.g., myoglobin) was very similar despite the very different three-dimensional fold; however, differences between all-α-helical globins and all-β-barrel Nbs suggest that nitrite reductase activity appears to be controlled by distal steric barriers, even though a more complex regulatory mechanism can be also envisaged.


Discussion
In order to have an overall view of the nitrite reductase activity of heme proteins and of their structural determinants, a list of heme proteins has been reported in Table 1. As a starting point, the second-order rate constant of the nitrite reductase activity of the heme proteins was compared to that of CO binding, which is usually considered as a probe of the energetic barriers (both on the distal and proximal side) for the reactivity of the heme-Fe(II) atom. Moreover, the coordination of the metal center in the ferrous form, which should be of some help in finding out the main determinants of the reactivity, has been reported. For the sake of consistency, only the values of the bimolecular rate constants for CO binding obtained by the rapid-mixing technique, which only can be employed for measurement of the nitrite reductase activity, have been reported. Accordingly, the values of the bimolecular rate constant for CO binding reported in Table 1, which were only obtained by flash and laser photolysis, such as for ferrous six-coordinated plant Hbs (i.e., Synechocystis Hb (S-Hb(II)), rice nonsymbiotic Hb(II) class 1, and Arabidopsis thaliana Hb (At-Hb(II) class 1 and class 2), have been calculated according to Equation (1) (see also footnotes to Table 1): where k bind is the intrinsic rate of CO binding (as observed by geminate recombination), k in and k out are the rates of ligand entry and exit, respectively, from the heme pocket, k diss is the dissociation rate of the endogenous six-coordinating ligand, and k ass is its association rate; therefore, k diss and k ass account for the partial six-coordination of the species [49]. Figure 5 shows the correlation between the nitrite reductase activity and the CObinding properties of heme proteins reported in Table 1. In the case of six-coordinated heme proteins, no apparent correlation was observed ( Figure 5, panel A), even though rice nonsymbiotic Hb class 1 displayed the fastest rate constants of CO-binding and nitrite reductase activity (Table 1); this suggests that in rice nonsymbiotic Hb class 1, the axial six-coordinating bond with the endogenous ligand represents a low free energy barrier for both exogenous ligands (i.e., CO and NO 2 − ). This occurrence might also be invoked for the relatively fast rate constants observed for other plant Hbs (i.e., At-Hb(II) class 1 and class 2 and S-Hb(II)), as compared to ferrous human neuroglobin (Hs-Ngb(II)) ( Figure 5, panel A, and Table 1). On the other hand, in the case of ferrous human cytoglobin (Hs-Cygb(II)), which in the S-S monomeric form shows a nitrite reductase activity about six-fold faster than At-Hb(II) class 2 (in spite of an almost 200-fold slower rate constant for CO binding, see Figure 5, panel A, and Table 1), a different functional modulatory behavior must be taken into account. Thus, in Hs-Cygb(II), the disulfide bond between CysB2 and CysE9 plays a dramatic role in modulating the nitrite reductase activity, and the reduction of the CysB2-CysE9 bond brought about a 50-fold decrease of the nitrite reductase activity, an effect much more marked than for CO binding, where only a 5-fold reduction was observed ( Figure 5, panel A, and Table 1). As a whole, in six-coordinated heme proteins, it looks like the His-Fe(II) axial sixth ligand regulates the barrier for CO binding, whereas in the case of nitrite reductase activity this is not the main determinant (as rice nonsymbiotic Hb(II) class 1 shows the fastest rate constant among all heme proteins investigated; see Table 1).
Conversely, in the case of five-coordinated heme proteins a linear correlation was observed between CO-binding rate constants and nitrite reductase activity ( Figure 5, panel B), suggesting that several structural features allow the discrimination between CO and NO 2 − . As shown in Figure 5 (panel B), at least four classes of heme proteins have been identified. They may differ for the discrimination between the two ligands, as represented by the displacement along the y-axis of the straight lines in Figure 5 (panel B). Thus, five-coordinated heme proteins have been classified according to Equation (2) as follows: Class I (straight red line in Figure 5, panel B) groups heme proteins, which strongly discriminate between the two ligands, being characterized by r ≥ 2.5 × 10 6 [i.e., the fastreacting form of ferrous Campylobacter jejuni truncated HbP (Cj-trHbP(II)), human serum heme-albumin (Hs-heme(II)-albumin), Equus ferus caballus cytochrome c complexed with cardiolipin (Efc-Cytc(II)-CL), Equus ferus caballus microperoxidase 11 (Efc-MP11(II)), the fast-reacting form of Methanosarcina acetivorans protoglobin (Ma-Pgb(II)), and tetrameric human Hb(II) (Hs-Hb(II)) in the R quaternary state].
Class IV (blue straight line in Figure 5, panel B) groups the slow-reacting form of Mt-trHbO(II) and all ferrous Nbs, which all showed a relatively poor discrimination power (r ≤ 2.5 × 10 4 ). duction of the CysB2-CysE9 bond brought about a 50-fold decrease of the nitrite reductase activity, an effect much more marked than for CO binding, where only a 5-fold reduction was observed ( Figure 5, panel A, and Table 1). As a whole, in six-coordinated heme proteins, it looks like the His-Fe(II) axial sixth ligand regulates the barrier for CO binding, whereas in the case of nitrite reductase activity this is not the main determinant (as rice nonsymbiotic Hb(II) class 1 shows the fastest rate constant among all heme proteins investigated; see Table 1).  The correlation emerging from the data in Figure 5 (panel B) indicated that within each class of heme proteins, a variation in CO-binding rate constant was accompanied by a similar behavior of the nitrite reductase activity, even though heme proteins belonging to different classes have a different way of discriminating between the two ligands. Thus, closely similar nitrite reductase activity between heme-proteins drastically differing for their CO binding behavior has been observed. A dramatic example is represented by tetrameric Hs-Hb(II) in the R-state, Pc-Mb(II) and Hs-Nb(II), which all displayed values of k on(NO2−)~6 .0 M −1 s −1 while values of the CO-binding rate constant greatly differed, spanning between 1.0 × 10 7 M −1 s −1 and 1.0 × 10 5 M −1 s −1 ( Figure 5, panel B, and Table 1). Although multiple conformations of both the distal and the proximal side of the heme pocket affect the CO-binding rate constant [79,80], a likely structural explanation has been attributed to the activation free energy for the ligand-induced movement of the Fe(II) atom into the heme plane, which is fairly low for tetrameric Hs-Hb(II) in the R-state, while it seems very high for Hs-Nb(II) [23,79]. As a matter of fact, it has been convincingly shown that a major contribution to the reactivity of CO with hemoproteins is represented by the energy required for the movement of the heme's Fe atom from its unliganded position (about 0.5Ǻ out of the heme plane on the proximal side) to the heme co-planar position in the CO-liganded form [2,79,81]; the major contribution stems from the steric repulsion between the imidazole of the proximal histidine and the heme pyrroles, which depends on the relative position and differs among various hemoproteins [2]. Obviously, the conformation of the distal portion of the heme pocket is also important to account for the different CO-binding behaviors shown by the various hemoproteins [80], but it is the variation of the activation free energy for the ligand-linked movement of the Fe-His bond which accounts for the modulation of the CO-binding kinetics of a specific protein by environmental conditions, such as pH [81].
On the basis of these considerations, class I may be representative of heme proteins with a very low activation free energy on both the proximal and the distal side of the heme pocket. It is illustrative that they also display a fairly fast nitrite reductase activity ( Figure 5, panel B, and Table 1); the slight variation within this class is due to small variations of the heme distal side conformation, affecting the kinetics for both ligands. On the other hand, class II and III appear to be heme proteins displaying a high proximal barrier for the reaction with CO, thus slowing down the carbonylation rate constant; however, this barrier does not affect dramatically the nitrite reductase activity. Therefore, differences in nitrite reductase activity within heme proteins belonging to class II and III is likely attributable to distal barriers. Lastly, class IV includes heme proteins (such as the slow-reacting form of the Mt-trHbO(II) and all ferrous Nbs), which have a very high free energy proximal barrier, which dramatically slows down the CO-binding rate constant. Consequently, differences in the nitrite reductase activity are probably due to a much higher distal barrier in Mt-trHbO(II) than in all ferrous Nbs, which display a very open heme pocket and a fairly fast nitrite reductase activity ( Figure 5, panel B, and Table 1). As a whole, different classes, reported in Figure 5 (panel B), reflect various free energy proximal barriers for CO-binding whereas different positions along the same line would refer to variations in the free energy distal barriers.
Even among ferrous six-coordinated heme proteins [i.e., Hs-Cygb(II) and Mus musculus Ngb(II) (Mm-Ngb(II)], the discriminatory power varied dramatically (r < 4.5 × 10 5 ; Figure 5, panel A, and Table 1). The low value of r depends on the low CO-binding rate constant, reflecting the occupancy of the sixth axial heme coordination by the heme distal histidyl residue and the strength of the axial Fe(II)-His distal bond. This is likely responsible for the variation in the nitrite reductase activity among the various six-coordinated heme proteins ( Figure 5, panel A, and Table 1). In particular, the longer the Fe-His proximal and distal bonds are in six-coordinated S-Hb(II), rice nonsymbiotic Hb(II) class 1, and At-Hb(II) class 1, as compared to bis-histidyl cytochromes, the lower the bond strength is. This is likely a factor enabling the heme distal His dissociation and the subsequent binding of exogenous ligands in six-coordinated Hbs [82]. Moreover, six-coordinated S-Hb(II), rice nonsymbiotic Hb(II) class 1, and At-Hb(II) class 1 displayed larger tilt angles for both the proximal and distal His residues compared with cytochrome b5. This decreases the strength of the heme-Fe-His bond contributing to fast ligand binding of these six-coordinated globins, likely playing a role of the utmost importance in characterizing their high nitrite reductase activity ( Figure 5, panel A, and Table 1) [82][83][84][85].
The kinetic and thermodynamic data were analyzed with the Prism 5.03 program (GraphPad Software, Inc., La Jolla, CA, USA). The results are given as mean values of at least four experiments plus or minus the corresponding standard deviation.

Conclusions
All Nbs show a fairly high nitrite reductase activity, a property which strengthens the hypothesis that they are mostly involved in the NO metabolism [18,20]. This enzymatic activity of heme proteins is one of the most efficient ways for the production of NO starting from the reduction of NO 2 − , a pivotal process for the regulation of blood vessel muscular tone and the regulation of the blood flow. Of note, in the retina, NO levels are crucial to maintain normal visual functions, being relevant for photoreceptor light transduction and the control of retinal blood flow, opening a perspective on a major role of Nbs in retinal disorders [88,89]. Moreover, a link between NO and Nb-based signaling and chemistry has been envisaged in M. tuberculosis, A. thaliana, D. rerio and H. sapiens. Specifically, the survival of M. tuberculosis in the host implies the presence of effective detoxification systems, including Nbs, to inactivate RNS and ROS produced by the immune response [19,25,26]. In A. thaliana, Nb has been hypothesized to transport and release NO at the infection site; moreover, NO may reduce the superoxide radical with the generating peroxynitrite that increases pathogen burden [16,19]. Interestingly, Dr-Nb may play a relevant physiological role in peroxynitrite scavenging from poorly oxygenated tissues, such as the retina in fish where blood circulation is critical for adaptation to diving conditions [21,22,26]. It is worth remarking that Danio rerio Nb shows the fastest nitrite reductase activity (Table 1), outlining the fact that, in fishes, the O 2 supply to poorly oxygenated tissues, such the retina, occurs by means of a fine regulation of the eye circulation through the rete mirabilis, with NO playing a major role in regulating the blood flow and thus the oxygenation of retinal layers [90]. Finally, Hs-Nb represents the C-terminal domain of the nuclear protein named THAP4, which displays a N-terminal modified zinc finger domain that binds DNA. Since Hs-Nb(III) binds NO without recognizing CO and O 2 , the Nb domain may be relevant for a NO-linked selective modulation of gene transcription [19,21,24].
Here, we report a comparison of a large number of heme proteins with drastically different conformations of the heme cavity, which casts light on the structure-function relationships, which modulate the nitrite reductase activity. In particular, we identified the accessibility of the heme distal pocket as an important factor since various heme proteins with different proximal constraints showed similar nitrite reductase activity. On the other hand, heme-proteins displaying different distal structural arrangements, but similar proximal constraints, show a remarkable effect on this enzymatic activity. However, other factors, such as the redox potential, cannot be discarded since the NO 2 − reduction to NO requires transient heme oxidation (see Scheme 1); unfortunately, for many of the investigated heme proteins this information is not yet available and a thorough comparison is presently not possible.